Project Summary The folate receptor is one of three major types of folate transporters. The human folate receptor is required for proper neural tube development, and is often overexpressed in cancers where it contributes to cancer progression. In both of these processes, there is emerging evidence that the foIate receptor acts independently of providing folates as vitamins for one-carbon metabolism. The small roundworm C. elegans obtains folates from its food source of bacteria. We have discovered that a specific bacterial folate (10-formyl-THF) increases the rate of proliferation of C. elegans germ stem cells, thereby acting as a signal to link the rate of proliferation to the availability of the animal?s bacterial diet. Additionally, we discovered that 10-formyl-THF acts as a signal to activate the NSM neurons. The activation of the NSM neurons causes starved animals to stop their forward motion when they encounter bacteria. Both of these processes require the C. elegans folate receptor, FOLR-1, and are independent of providing folates for their canonical role in one-carbon metabolism. The goal of this proposal is to identify the molecular pathways through which FOLR-1 mediates signals from the stimulatory folate to germ cells and the NSM neurons. We will use fluorescent tags to determine where FOLR-1 is localized in cells and whether the localization changes in the presence of stimulatory folates. Genetic experiments suggest that FOLR-1 functions in both neuronal and non- neuronal tissues to mediate the activation of the NSM neuron. We discovered that the GON-2 calcium channel is required for the calcium influx that accompanies NSM neuronal activation. Similar to FOLR-1, GON-2 is also required in both neuronal and non-neuronal tissues for NSM activation. Using genetic approaches, we will identify the tissues in which FOLR-1 and GON-2 each function to allow NSM activation. We discovered that GON-2 is also required both to maintain calcium levels in the germline and for normal germ cell proliferation, suggesting that increased calcium levels promote germ cell proliferation. We will determine if calcium levels differ in response to growth conditions and stimulatory folate, and the role of GON-2 and FOLR-1 in altering calcium levels. Genetic approaches will be used to identify the downstream effectors that alter germ cell proliferation in response to altered calcium levels in the germline. Unbiased genetic screens will be used to identify components and regulators of the stimulatory-folate pathway(s). The identified genes will be analyzed using genetic and biochemical approaches to determine how they contribute to the pathway(s). This research will uncover the molecular pathway(s) through which folates regulate diverse cellular processes independently of their role in metabolism. This will provide a paradigm for understanding non-canonical folate pathways, with the potential for insights into neural tube defects and cancer.